Binge drinking, or the consumption of a large volume of alcoholic beverages in a very short time, is a serious social problem, particularly among adolescents and young adults. Ethanol (EtOH), the main ingredient in alcoholic beverages, is well known for its behavioral and psychological effects and also as an immune system modulator. However, the underlying mechanisms by which EtOH exerts its various effects are still not defined. Although recent evidence indicates that EtOH acts at the cellular level, there i still a fundamental gap between EtOH's potential cellular targets and its subsequent physiological effects. One such target is a group of cell membrane ion channels called transient receptor potential (TRP) channels. TRP channels are found in various cell types, including brain microvascular endothelial cells (BMVEC) of the blood-brain barrier (BBB). TRP channels mediate certain immune responses, such as cytokine production and leukocyte-endothelial adhesion (LEA), the initial step in the inflammatory process. Activation of TRP channels by various extracellular stimuli, including EtOH, induces an influx of calcium ions, which can subsequently increase LEA. Alcoholic beverages differ in their EtOH content or alcohol-by-volume (ABV) concentration, and EtOH's effects appear to be concentration dependent. We recently reported that binge consumption of solutions with high EtOH concentrations causes more pronounced immune responses than those with low EtOH concentrations, even when the amount of EtOH intake is the same. Based on the recent literature and our preliminary studies, we hypothesize that TRP channels mediate alcohol- induced immune responses at the blood-brain barrier (BBB) in an EtOH concentration- dependent manner. To test our hypothesis, we propose the following two specific aims: (1) To determine the effects of EtOH concentration on the structural binding of EtOH to TRPV4 channels in BMVEC using NMR spectroscopic techniques; and (2) To delineate the involvement of TRPV4 channels in the EtOH concentration-dependent effects on immune responses in the BMVEC at the BBB. In this application, we will combine investigation of the structural biology of EtOH-protein interactions with examination of EtOH's effects in animal and cell culture models to determine the mechanisms by which EtOH affects immune responses. This study is innovative because, to our knowledge, few studies have examined the relationship between EtOH-protein interaction and EtOH-induced immune effects at the level of the vascular endothelium. The NMR structural studies will provide essential details concerning EtOH-TRP binding and the EtOH concentration-dependent changes in target protein conformation. The molecular studies will then determine the correlation between those changes and the immunomodulatory effects of EtOH. Our study is highly significant and clinically relevant because it will provide valuable information on the mechanisms underlying the physiological effects of binge drinking with high ABV alcoholic beverages, which can help to prevent EtOH-induced dysregulation of immune responses and be used to develop therapeutic strategies to treat patients with alcoholic intoxication seen in the emergency room.